Bacteriologic testing method and means therefor



April 12, 1966 R. GUTHRIE 3,245,882

BACTERIOLOGIC TESTING METHOD AND MEANS THEREFOR Filed A ril 16, 1962 2 Sheets-Sheet l UNKNOWN 2m 4mg 8mg lZmg ZOmg /fl\ E7 Z 6 o 0 o o 0 UNKNOWN 2mg 4mg 8mg. lZmg 20mg INVENT ROBERT 607/79? SM (a. M

ATTORNEY April 12, 1966 R. GUTHRIE 3,245,882

BACTERIOLOGIC TESTING METHOD AND MEANS THEREFOR Filed April 16, 1962 2 Sheets-Sheet 2 UNKNOWN:

IN V EN TOR.

ROBf/lf GUT/WW5 A TZORNEY United States Patent 3,245,882 BACTERIOLOGIC TESTING METHOD AND MEANS THEREFOR Robert Guthrie, Williamsville, N.Y., assignor to the United States of America as represented by the Secretar-y of Health, Education, and Welfare Filed Apr. 16, 1962, Ser. No. 187,707 16 Claims. (Cl. 195-103.5)

This invention relates to a bacteriologic method and means for use in the qualitative detection and quantitative estimation of phenylalanine blood levels.

The importance and usefulness of diagnostic tests for the detection and estimation of phenylalanine in body fluids is well known to the medical practitioner, particularly in phenylketonuria, a rare inherited methabolic abnormality, occurring once in every 10,00020,000 births. Phenylketonuria, more commonly known as PKU, is a condition wherein the body is unable to utilize an essential amino acid, phenylalanine, at the normal rate. Normally, phenylalanine is metabolized in the liver to tyrosine by the action of the enzyme, phenylalanine hydroxylase. In the phenylketonuric patient, however, this enzyme activity in the liver is missing through inheritance. Thus, when phenylalanine is ingested, instead of being normally metabolized, it increases in abnormal amounts in the bloodstream along with the subsequent spillage of by-products, principally phenylpyruvic acid, into the urine. Normally, the blood phenylalanine level in an individual is between 1 and 4 mg. per 100 ml., whereas in the phenylketonuric patient it may be as high as 60 mg. per 100 ml. It appears that the resulting mental retardation in the phenylketonuric patient is due to either directly or indirectly to this high phenylalanine level or to the associated metabolic products of the phenylalanine.

When PKU is present and detected early in life, the infant has the opportunity for a normal or near-normal life when treated with a special diet low in phenylalanine content. When PKU is present and not detected, the infant faces a lifetime of mental deficiency usually sufficiently severe to require institutional dependency.

Since the discovery of PKU by Polling in 1935, a simple ferric chloride test for phenylpyruvic acid in urine has been the standard method of detecting this disease. With infants, however, the test may not reliably be performed until after about 4 weeks of age, at which time detectable amounts of phenylpyruvic acid appear in the urine. The test consists of placing a drop of percent aqueous solution of ferric chloride on the infants most recently wet diaper. A positive reaction is the immedi ate appearance of a blue-green or gray-green color, which indicates the presence of phenylpyruvic acid in the urine. The ferric chloride test, however, is subject to several serious limitations. These include (1) failure of young infants to produce a positive test until several weeks after birth, even though there is evidence that the blood phenylalanine concentration increases rapidly in the first few days of life in PKU conditions and (2) failure of some older PKU cases to produce a positive ferric chloride test if blood phenylalanine levels are below approximately mg. percent.

These limitations have been overcome by the methods and means of this invention for detecting elevated phenylalanine blood levels, particularly in the 4 to 20 mg. percent range. The invention may be applied to blood specimens taken from new born infants even as early as the fourth or fifth day after birth.

It is, therefore, an object of the present invention to provide a bacteriologic method for detecting the presence of phenylalanine in blood.

It is also an object of the present invention to provide methods and means for qualitatively and quantitatively determining phenylalanine blood levels.

Another object of this invention is to provide bacteriologic methods and [means for the qualitative detection and quantitative estimation of abnormal concentrations of phenylananine in blood.

A further object is to provide a bacteriologic test for the qualitative and quantitative determination of phenylalanine levels in blood specimens taken from infants as early as the fourth or fifth day after birth.

An additional object is the provision of a reliable test for detecting elevated phenylalanine blood levels, particularly in the 4 to 20 mg. percent range.

Still another object is to provide a process for preparing a simple testing device for effecting an accurate determination of blood phenylalanine levels.

Still a further object is to provide a testing means by which phenylalanine blood levels may be qualitatively and quantitatively determined.

These and other objects of the present invention will become apparent as the detailed description thereof proceeds.

According to the present invention, a particular bacterial metabolism is utilized for a specific diagnostic purpose. The basis of the invention resides in what is conveniently called an inhibition assay. In general terms, a suitable bacterial microorganism is inhibited from growing in a culture medium by the addition of an antimetabolite that normally inhibits its growth. This inhibition, however, is reversed in the presence of phenylalanine, an antagonist to the inhibitory agent, and the bacteria grow. If, therefore, the growth-inhibited bacteria are contacted with a blood specimen containing phenylalanine, the resultant growth of said bacteria qualitatively indicates the presence of phenylalanine therein. To determine quantitatively the amount of of phenylalanine in the blood specimen, the intensity of growth observed, which is proportional to the amount of phenylalanine that is present, is compared with the growth of controls constituting known concentrations of phenylalanine.

The foregoing generalization may be exemplified by an agar diffusion technique employing spores of Bacillus subtilis (American Type Culture Collection, Strain 6051), hereinafter referred to as B. subtilis, as the bacterial microorganism. Utilizing the usual laboratory methods, a minimal sugar-salts agar medium is prepared. With B. subtz'lis, I have found a modified Demains Medium (J. Bact., 75, 517-522, 1958), to be particularly suitable for the purpose of this invention. The medium is supplemented with 2 10- M meta-Z-thienylalanine, a phenylalanine analog, which produces a growth inhibition in B. subtilis that is relieved by phenylalanine. While the agar medium is in a somewhat cooled but liquid state, circa 50-55 C., B. sublilis spores are inoculated therein. The liquid culture is then poured out into a suitable shallow receptacle, such as a Petri dish or a Pyrex baking dish, and allowed to harden.

Discs, approximately one-quarter inch in diameter, are cut from a sheet of suitable filter paper, thoroughly impregnated with a series of standard phenylalanine solutions of appropriate concentrations and dried. In commercial production, these standards are cut or stamped from sheets of filter .paper following impregnation. Similar sized discs are impregnated with blood samples to be analyzed.

The assay is carried out by placing the blood impregnated discs (i.e., the Unknown) and the discs impregnated with known concentrations of phenylalanine (i.e., the Controls) on the surface of the medium and the receptacle is incubated, usually at 25 30 C. During the period of incubation, the phenylalanine in the Control discs and, if present, in the Unknown discs diffuses out into the medium. As the diffusing phenylalanine comes 3 in contact with the growth-inhibited B. subtilis, the inhibition due to beta-2thienylalanine is reversed and turbid, circular, growth zones of B. sablilis are formed around the discs.

-In general, the appearance of a growth zone surrounding an Unknown disc qualitatively indicates the presenee of phenylalanine in the corresponding blood specimen and the diameter of the growth zone quantitatively indicates the concentration of phenylalanine there-in; the greater the zone of growth, the greater the concentration of phenylalanine. By simply comparing the diameter of .the growth zones surrounding the Unknown discs with those surrounding the Control discs, one can estimate the corresponding phenylalanine level of the blood samples. In turn, the absence of a growth zone around an Unknown disc illustrates a complete lack of .phenylalanine in the corresponding blood sample. My invention, therefore, may be utilized to determine both a-bnormally high and abnormally low phenylalanine blood levels.

l have also found that the foregoing principles may be conveniently utilized in a simple testing device comprising an essentially fiat piece of absorbent material impregnated with a growth-inhibited bacterial microorganism, which inhibition is reversed by phenylalanine, and a growth-promoting medium therefor. After moistening the test device with Water, the previously described Cont-rol discs and Unknown discs are placed in contact with the surface of the device and the assembly is incubated. After incubation, the resulting bacterial growth zones surrounding the discs are similarly compared.

This testing device is illustrated more or less diagrammatically in the accompanying drawing wherein FIGURE 1 is a plan view of one form of my invention;

FIGURE 2 is a plan view of a variant form of my invention; and

FIGURE 3 is a plan view of another variant form of my invention.

To illustrate herein a preferred embodiment of this invention, I have used the bacterial strain, Bacillus sabtilis (ATCC-6051), inhibited with beta-Z-thienylalanine as one inhibition assay. The methods and means of this invent-ion, however, are not limited to this particular bacterium nor to this particular antimetabolite. Any strain of bacteria whose growth is inhibited by an antimetabolite may be employed in the test system so long as the growth inhibition caused by the antimetabolite is reversed in the presence of phenylalanine. For example, Escherichia coli, inhibited with DL-para-fluo-r-ophenylalanine, has also been found suitable. Because of the enormous numbers of potential antimetabolites produced by the large cancer chemotherapy organic synthetic programs during recent years, and because of the many diverse microorganisms available, the list of possible inhibition assays that are specific for phenylalanine, using a microbe inhibited by an antimetabolite as a test system, appears almost endless.

Exemplifying a more detailed description of the invention is the following:

Preparation of blood specimens to be analyzed A small amount of [fresh blood obtained by skin puncture is applied immediately to a piece of thick filter paper, such as, for example, Schleicher & Schuell #903 or Whatman #31-ET, and dried. The amount of blood required should be sufiicient to thoroughly soak through the filter paper at the point of contact and the blood spot, when air-dried, should be at least one-half inch in diameter and close enough to the edge of the paper so as to facilitate cutting or stamping out a disc with a paper punch. Tlhe filter paper is preferably steamed or autoclaved at 15 pounds pressure for 5 minutes to coagulate blood proteins and to prevent blood pigments from diffusing from the paper discs into the culture medium later during incubation, thereby masking possible growth zones. A disc, approximately one-quarter inch in diameter, is then cut or punched (e.g. with a A punch) from the blood spot and is ready for assay as an Unknown. Fine-tipped forceps are recommended in handling the blood-impregnated discs.

The small sample of blood required (approximately 0.01 ml. per quarter inch disc) makes it possible to use blood obtained from a heel puncture of newly born infants. This permits daily blood assays, if desired, even in small infants.

Preparation of standard phenylalanine discs Aqueous solutions of L-phenylalanine are prepared with resulting concentrations of, for example, 2, 4, 6, 8, 10, 12 and 20 mg. per 100 ml. These concentrations are quite suitable for routine screen of infants to determine possible abnormal blood phenylalanine levels in the early weeks of life. Higher concentrations, such as from 20 to '60 mg. per 100 ml., can be included as, for example, when analyzing blood samples from older PKU patients. One-quarter inch discs of filter paper, of the same type that is used in preparing the blood specimens, are impregnated with 0.01 ml. each of the standard phenylalanine solutions, dried, and stored, preferably under refrigeration, until needed. Alternatively, large sheets of the same type filter paper may be immersed in each solution until thoroughly soaked, drained and dried. When so prepared, these large sheets, which may be stored in airtight containers for long periods of time, offer a longlasting supply of Controls for the assay technician, since only small-sized discs cut from these sheets are thereafter required for use in the assay procedure.

Control discs may also be prepared from normal blood to which has been added known concentrations of L-phenylalanine. In this case, however, the sheets of filter paper, after impregnation, must be autoclaved as was done with the Unknown blood specimens.

In practice, I have found extremely little dilference between the two and recommend, for convenience, the use of the former.

Preparation of Demains Medium (modified) for spare The above ingredients are dissolved in and diluted with distilled water to 900 ml. (pH=6.8-7.0). milliliters are dispensed into each of 10 suitable bottles (e.g., 8 ounce prescription bottles) and sterilized. The Salt Solution incorporated into the above recipe is an aqueous solution of the following salts:

G./l. MgSO 7H O 10.0 MI'1C124H2O F6C13 CaCl 0.5

10 milliliters of a separately sterilized 10% dextrose solution is added to each bottle just before use in the assay to make ml. of a sugar-salts medium.

Procedure for growing spores of Bacillus subtilis Raw potatoes are chopped up and Weighed. Enough water is added to cover the chopped potatoes and they are cooked until soft and friable. After cooking, the water is drained off, the potatoes strained through several layers of cheese cloth and then diluted with water to 10 times their original weight. One and one-half percent of Difco Bacto-agar (plain, no nutrient) and one-tenth percent of yeast extract are added. The mixture is brought to a boil, cooled to about 50 -55 C., and heavily inoculated with B. subtilis spores in preparing slants (18 x 150 mm. tube) which are then incubated at 30- 37 for 4-5 days.

Procedure for preparing B. subtilis spore suspension With a glass rod, the bacterial growth is scraped off the agar surface of the slant, washed off with ml. of 0.9% NaCl into a centrifuge tube and centrifuged. The supernatant liquid is decanted oif and the spores are rewashed and centrifuged three or four times with fresh 10 ml. portions of 0.9% NaCl. The final spore suspension is made by adding sufiicient distilled water to yield an optical density of 0.9 .at 550 m wave length on a colorimeter. This suspension may be kept refrigerated at 2-5 C. for several months to a year. In practice, 0.2 ml. of this suspension is used per 200 ml. of Demains Agar Medium. Alternatively, 0.2 ml. of this suspension may be added to each of several suitable vials. After the water has evaporated, the dried spores may be maintained indefinitely without refrigeration until needed, the spore contents of each vial being sufiicient for 200 ml. of Demains Agar Medium.

Assay procedure A bottle containing 100 ml. of a sugar-salts medium is placed in a 55 C. water bath. A bottle containing 100 ml. of 3% agar is placed in boiling water until completely melted and then placed in the 55 C. water bath. 0.4 milliliter of M/ 100 beta-Z-thienylalanine solution and 0.2 ml. of B. subtilis spore suspension are added to the medium bottle which is in turn quickly poured into the bottle containing the 3% melted agar and thoroughly mixed together. The contents are then emptied out into a suitable shallow dish. This step is completed as quickly as possible to minimize exposure of the bacteria to elevated temperatures. The above quantities will adequately cover an 8" x 12" Pyrex baking dish which is quite suitable for running an assay on approximately 40 Unknowns at one time.

When the agar medium has solidified, the discs impregnated with the phenylalanine Controls" and the discs impregnated with the Unknwon blood samples are placed in rows on the agar surface. The dish is then incubated, preferably at 25 30 C. A second dish can be inverted over the agar dish as a cover or a sheet of aluminum foil can be used. After incubation, one simply compares the diameter of the turbid growth zones around the Unknown discs with those around the Control discs and by inspection one can select the Control containing the corresponding phenylalanine level with an accuracy of plus or minus 2 mg. percent.

Growth zones are often visible within 6-8 hours. For best results, however, the results should be read after 16-24 hours incubation. After 30 hours, increasing background growth in the agar may interfere with an accurate interpretation of the results.

The term incubation is utilized in its ordinary sense in bacteriology, meaning the maintenance of an inoculated medium under conditions such as would normally result in growth and proliferation of the microorganism, assuming the presence of all necessary nutrients for the microorganism in the medium.

Test device The testing device of this invention comprises an essentially flat piece of absorbent material impregnated with a medium containing a growth-inhibited bacterial microorganism, which inhibition is reversed in the presence of phenylalanine. In the preferred embodiment, suitably 1 California Corporation for Biochemical Research, Catalog No. 5901 kept frozen until needed.

6 spaced areas are marked on the absorbent material to indicate the positioning of the Control and Unknown discs to be used with the device.

Referring to FIGURES 1, 2 and 3, various forms of testing devices which may be employed according to this invention are illustrated comprising an essentially flat absorbent material 10 having a series of spaced circular areas 11 of equal diameter marked thereon. Said circular areas are preferably spaced at least one inch apart and are preferably one-quarter inch in diameter. A plurality of circles 12, outwardly projecting from each of said circular areas and having a common center with said circular areas and with each other may also be marked on said absorbent material (not shown in FIG- URE 2). The distance between said circles is preferably l-2 mm. Holding means 13, consisting of a nonporous material, such as heavy cellophane, almuinum metal foil or a firm plastic, and secured to one or both ends (not shown) of said absorbent material, may be provided although such is not an essential component of my testing device.

The preparation of the test device in accordance with this invention involves simply impregnating the absorbent material 10 with a liquid culture medium containing a bacterial microorganism whose growth has been inhibited by the presence of a suitable antimetabolite in the medium. Any liquid medium, which is suflicient in all nutrients for the selected strain of bacteria to grow therein may be employed. A particularly suitable culture comprises the heretofore mentionad B. subtilis spores in a modified Demains Medium, supplemented with 2 10 M beta-Z-thienylalanine as the B. subtilis growthinhibitor. Agar may or may not be added to the culture medium and, if added, the impregnation of the absorbent material should take place after the agar has first been liquefied and cooled to 50-55 C. After the absorbent material is impregnated with the culture, it is subject to drying either at or near room temperature and at atmospheric pressure, and preferably in a current of air (30-40 C.) to hasten the drying. For storing over extended periods of time, the testing device should be sealed in sterile, moisture-free containers or envelopes.

The absorbent material employed in this invention may be any material of a porous nature, such as thick filter paper, matted cotton, cloth, or other bibulous material which comprises a mass or complex of cellulose fibers, so long as it will readily absorb or otherwise pick up a substantially constant amount of the impregnating culture, so as to permeate substantially throughout the thickness thereof as distinguished from mere surface application, and is capable of retaining said culture when later moistened for use. The thickness of the absorbent material should be such as to hold a suflicient amount of the culture medium therein for the subsequent growth of the bacteria.

In use, the testing device of this invention is saturated with water, e.g., by quickly immersing the device into a container of water until thoroughly moistened, and placed on a flat surface, care being taken not to excessively soak the device, so that the culture impregnated therein is not lost through diffusion, dilution or some other mechanism. One-quarter inch discs of filter paper, impregnated with a series of standard phenylalanine solutions of appropriate concentrations (i.e., the Controls) are prepared in the same manner as described heretofore and placed in contact with a suitable number of the marked circular areas 11 on the testing device. For example, as illustrated in the device of FIGURE 1, five circular areas have been marked for Control discs impregnated with phenylalanine solutions of 2, 4, 8, 12 and 20 mg. percent. A disc, impregnated with the blood sample to be analyzed (i.e., the Unknown) is placed in contact with another circular area on the testing device. The entire assembly is then incubated, preferably at 25 -30 C. for 16-24 hours. During the period of incubation, the

phenylalanine in the discs diffuses out into the mediumimpregnated absorbent material and comes in contact with the growth-inhibited bacterial microorganism absorbed therein, thereby causing the inhibition to be reversed. Throughout incubation, the device should be maintained in a moistened condition to provide an optimum medium for supporting the resulting growth of the bacteria. After incubation, the device is treated with a suitable bacterial stain .in the conventional manner'to color the resulting growth zones of bacteria surrounding the discs. By then comparing the diameter of the growth zone, if any, surrounding the Unknown disc with those surrounding the Control discs, the qualitative and quantitative determination of phenylalanine in the analyzed blood sample is obtained.

To facilitates this comparison, suitably spaced circles, superimposed about the disc area, are preferably provided on the testing device as illustrated in FIGURES 1 and 3, thereby providing a self-contained measuring means in the device. One need simply compare the number of circles engulfed by the growth zone surrounding the Unknown disc with the number engulfed about the Controls and, in a matter of seconds, estimate the corresponding phenylalanine concentration in the analyzed blood sample. The presence of these circles, however, on the testing devices of this invention is not critical and may be omitted, as shown in FIGURE 2, in which case, one must then employ external measuring means for measuring the diameters of the growth zones.

Testing devices, adapted to accommodate an assay for the determination of phenylalanine blood levels in multiple blood specimens, are also contemplated as constituting part of this invention. Furthermore, there is no restriction on the number of, or concentration of, the phenylalanine Control discs that may be used. For example, referring to FIGURE 3, a test device is illustrated wherein six Unknown disc areas are provided for, as indicated by the letters A, B, C, D, E and F, for analysis with Controls constituting 2, 4, 6, 8, 12 and 20 mg. percent phenylalanine concentrations. Higher concentrations, however, could easily be used as, for example, between the 20-60 mg. range when analyzing blood samples obtained from older PKU patients. Although the testing device of this invention is depicted in the accompanying figures as an essentially flat strip of absorbent material, any other physical embodiment such as a continuous roll or large sheets, which could accommodate fifty or a hundred Unknown discs at one time, may also be used.

Discussion From the foregoing considerations, the bacteriologic test, or inhibition assay, of this invention is deemed a reliable diagnostic tool for the detection of phenylalanine in blood and particularly for use in the estimation of abnormal concentrations of phenylalanine therein. The limitations inherent in the conventional ferric chlorideurine test for phenylpyruvic acid have been overcome. By utilizing the method and means herein, one can readily detect elevated phenylalanine blood levels in new born infants, particularly in the 42() mg. percent range that generally escapes detection with the ferric chloride test.

Furthermore, this invention is simple enough to be used in mass screening programs of infants for the detection of phenylketonuria. Such screening programs are particularly within the capabilities of most hospital laboratories. As late as practical before discharge from the hospital and preferably not earlier than the third day of life, the infants heel is punctured, blood is spotted on a filter paper and allowed to dry. At weekly intervals, all filter papers collected are subjected to assay. A screening program on a community level may also be conducted by the submission of blood-impregnated filter papers from all local hospitals and physicians within the community to. a central laboratory. At least once a week,

accumulated filter papers are steamed,'paper discs are punched out from each blood spot, and subjected to assay. It is estimated that a single technician may be able to test 500 specimens per week.

For rapid on-the-spot assays, the testing devices de scribed herein are particularly suited. These devices may be easily prepared in advance in large numbers, are stable under dry, sterile conditions for long periods of time, and can be used when needed with a minimum of effort. With these devices and a suitable series of standard phenylalanine discs, large numbers of which can also be prepared in advance and used from storage as needed, the inhibition assay of this invention is adapted for use within the physicians own office.

While the foregoing description is directed to the preferred operational methods and means of the present invention, it will be apparent that certain procedural details as herein described may be modified by one skilled in the art without departing from the substance of the actual invention; and it will be understood, therefore, that it is intended and desired to embrace within the scope of the invention such modifications and changes as may be desirable or necessary to adapt the invention to varying conditions and uses as defined by the scope of the appended claims.

Having thus described my invention, what I claim and desire to protect by Letters Patent is:

1. The method of visually detecting the presence of phenylalanine in blood which comprises contacting with the blood to be tested a medium containing a bacterial microorganism and a growth inhibitor effective to prevent the growth of said microorganism and which can be rendered ineffective by contact with phenylalanine, and incubating said microorganism in the presence of said medium and said blood, whereby subsequent growth of said microorganism is indicative of the presence of phenylalanine in said blood.

2. The method of claim 1 wherein the proteins in said blood are coagulated prior to contact of said blood with said medium.

3. The method of claim 1 wherein the bacterial microorganism is a strain of Bacillus subtz'lis.

4. The method of claim 1 wherein the microorganism is Bacillus subtilis and the growth inhibitor is beta-2- thienylalanine.

5. The method of visually determining blood phenylalanine level which comprises placing a first disc of bibulous material impregnated with the blood to be tested and a plurality of similar second discs of identical size impregnated respectively with known different concentrations of phenylalanine in contact with a medium containing a bacterial microorganism and a growth inhibitor effective to prevent the growth of said microorganism and which can be rendered ineffective by contact with phenylalanine, incubating said medium in contact with said discs, and thereafter visually comparing any growth zone of said microorganism at said first disc with those at said second discs.

6. The method of claim 5 wherein prior to contact of said first disc with said medium the proteins in said blood are coagulated by exposure of said blood impregnated bibulous material to steam.

7. The method of claim 5 wherein said bacterial microorganism is Bacillus subtilis and said growth inhibitor is beta-Z-thienylalanine.

8. For use in the visual determination of blood phenylalanine level, the combination of a growth-promoting medium, a bacterial microorganism in spore form in said medium, and a growth inhibitor in said medium effective to prevent growth of said microorganism and which can be rendered ineffective by contact with phenylalanine.

9. The combination of claim 8 wherein said bacterial microorganism is Bacillus subtilis and said growth inhibitor is beta-Z-thienylalanine.

10. A device for visual testing comprising a fiat piece of absorbent material impregnated with a growth-promoting medium containing a bacterial microorganism in spore form and a growth inhibitor effective to prevent the growth of said microorganism and which can be rendered ineffective by contact with phenylalanine.

11. A device for visual testing comprising a member having a flat surface portion formed of absorbent material impregnated with growth-promoting agent, a bacterial microorganism in spore form and a growth inhibitor effective to prevent the growth of said microorganism and which can be rendered ineffective by contact with phenylalanine.

12. The device of claim 11 in which said microorganism is Bacillus subtilis and said growth inhibitor is beta-Z-thienylalanine.

13. A testing device according to claim 11 wherein said flat surface of said member bears indicia in the form of spaced apart identical first and second circles having identical concentric circles of larger diameter in surrounding relation therewith, said first and second circles indicating contact areas for discs of like diameter bearing blood to be tested and bearing known different concentrations of phenylalanine respectively, said concentric circles affording a measure of the comparative size of the zones of growth of said bacterial microorganisms resulting from placement of such discs in contact with said surface.

14. A testing device according to claim 11 wherein said bacterial microorganism is Bacillus subtilis, said growth inhibitor is beta-Z-thienylalanine and said flat surface bears indicia in the form of spaced apart identical first and second circles having identical concentric circles of larger diameter in surrounding relation therewith, said first and second circles indicating contact areas for discs of like diameter bearing blood to be tested and bearing known different concentrations of phenylalanine respectively, and said concentric circles affording a measure of the comparative size of the zones of growth of said Bacillus subtilus resulting from placement of such discs in contact with said surface.

15. A testing device for visually determining blood phenylalanine level comprising a carrier of absorbent material having absorbed therein a liquid growth-promoting medium containing a bacterial microorganism in spore form, and a growth inhibitor elfective to prevent the growth of said microorganism and which can be rendered ineifectively by contact with phenylalanine, said carrier having a fiat surface provided with a plurality of spaced circles of,equal diameter marked thereon, a first disc of absorbent sheet material having a diameter equal to that of said circles impregnated with the blood to be tested and overlaying said carrier surface in registry with one of said circles, and a plurality of second discs similar to said first disc impregnated with known different concentrations of phenylalanine respectively and each overlaying said carrier surface in registry with a different one of said circles.

16. A device for visual testing comprising a bibulous carrier impregnated with the dried residue of a liquid growth-promoting medium having incorporated therein a bacterial microorganism in spore form and a growthinhibitor effective to prevent the growth of said microorganism and which can be rendered ineifective by contact with phenylalanine.

References Cited by the Examiner UNITED STATES PATENTS 4/1960 Rdzok et al. 103.5

OTHER REFERENCES A. LOUIS MONACELL, Primary Examiner.

ABRAHAM H. WINKELSTEIN, Examiner.

A. E. TANENHOLTZ, Assistant Examiner. 

1. THE METHOD OF VISUALLY DETECTING THE PRESENCE OF PHENYLALANINE IN BLOOD WHICH COMPRISES CONTACTING WITH THE BLOOD TO BE TESTED A MEDIUM CONTAINING A BACTERIAL MICROORGANISM AND A GROWTH INHIBITOR EFFECTIVE TO PREVENT THE GROWTH OF SAID MICROORGANISM AND WHICH CAN BE RENDERED INEFFECTIVE BY CONTACT WITH PHENYLALANINE, AND INCURBATING SAID MICROORGANISM IN THE PRESENCE OF SAID MEDIUM AND SAID BLOOD, WHEREBY SUBSEQUENT GROWTH OF SAID MICROORGANISM IS INDICATIVE OF THE PRESENCE OF PHENYLALANINE IN SAID BLOOD. 